Nanoplastics Accumulate in Mouse Testes and Trigger Sperm-Killing Ferroptosis via CISD1
50 nm polystyrene nanoplastics accumulate in mouse testes, degrading sperm quality and triggering iron-dependent cell death through a newly identified mitochondrial pathway.
Summary
Researchers exposed male mice to environmentally relevant doses of 50 nm polystyrene nanoplastics (PS-NPs) for 35 days and found significant testicular damage and reduced sperm quality. PS-NPs accumulated in lysosomes of spermatocytes, then migrated to mitochondria, flooding them with ferrous iron and reactive oxygen species. This triggered ferroptosis — an iron-dependent form of programmed cell death — partly by suppressing CISD1, a mitochondrial protein that normally limits iron uptake into mitochondria. Blocking autophagy or chelating iron reduced cell death. The diabetes drug pioglitazone, which stabilizes CISD1, also mitigated ferroptosis, pointing to a potential therapeutic target for nanoplastic-induced male infertility.
Detailed Summary
Nanoplastics are everywhere — in our food, water, air, and now confirmed in human blood, placenta, and testes. Polystyrene nanoplastics (PS-NPs) are among the most prevalent types detected in human testicular tissue, yet the molecular mechanisms by which they damage male reproductive health have remained poorly understood. This study from Northwest A&F University provides the most mechanistically detailed account to date of how PS-NPs harm spermatocytes, linking nanoplastic exposure to ferroptosis — an iron-dependent, lipid peroxidation-driven form of programmed cell death — through a novel ferritinophagy–CISD1 axis.
In the animal arm of the study, twenty male KM mice (5 weeks old) were randomly divided into control and PS-NP groups (n=10 each). The PS-NP group received 50 mg/kg of 50 nm PS-NPs daily by oral gavage for 35 days, a dose calculated to fall within environmentally relevant human exposure ranges using body surface area conversion. Pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) confirmed PS-NP accumulation in testicular tissue. Histological analysis using the Johnsen scoring system revealed disrupted seminiferous tubule architecture, and CASA-based sperm analysis showed significantly reduced sperm density, motility, and viability, alongside elevated abnormality rates. Serum testosterone and FSH levels were also altered, pointing to disruption of the hypothalamic–pituitary–gonadal axis.
In parallel, mouse spermatocyte GC-2 cells were exposed to 50, 100, and 200 µg/mL PS-NPs. Cell viability declined in a dose-dependent manner, confirmed by CCK-8 and Calcein/PI live-dead assays. Ferroptosis markers were robustly elevated: intracellular ferrous iron (Fe²⁺) increased, MDA levels rose, GSH was depleted, lipid peroxidation (quantified by C11-BODIPY 581/591 fluorescence and flow cytometry) surged, and ferroptosis-regulating proteins including GPX4, SLC7A11, and FTH1 were downregulated. Crucially, treatment with deferiprone (DFP, an iron chelator, 3.5 µM) and 3-methyladenine (3-MA, an autophagy inhibitor, 10 mM) both significantly reduced ferroptosis markers, establishing that iron overload and autophagic flux are required for PS-NP-induced cell death.
Time-course fluorescence microscopy using Cy5-labeled PS-NPs tracked the intracellular journey of the particles over 0–12 hours. PS-NPs first concentrated in lysosomes, then transferred to mitochondria. This lysosome-to-mitochondria trafficking coincided with increased mitochondrial Fe²⁺ and mitochondrial ROS, structural mitochondrial damage (confirmed by electron microscopy showing condensed cristae and outer membrane rupture), and a marked decrease in CISD1 — a mitochondrial outer membrane iron-sulfur protein that normally restricts ferrous iron entry into the mitochondrial matrix. Simultaneously, NCOA4 (the selective autophagy receptor for ferritin) was upregulated, driving ferritinophagy: autophagic degradation of ferritin heavy chain 1 (FTH1) in lysosomes, releasing free Fe²⁺ into the cytoplasm and subsequently into mitochondria.
Overexpression of NCOA4 in GC-2 cells replicated the ferroptotic phenotype, while intervention with pioglitazone (5 µM) — a thiazolidinedione diabetes drug known to stabilize CISD1 protein — significantly rescued CISD1 expression, reduced mitochondrial iron overload and ROS, restored mitochondrial membrane integrity, and attenuated ferroptosis markers. This identifies the NCOA4-ferritinophagy → Fe²⁺ release → CISD1 loss → mitochondrial iron flooding pathway as the central mechanism of PS-NP reproductive toxicity, and pioglitazone as a candidate protective agent. The study is among the first to mechanistically link ferritinophagy to CISD1 suppression in any cell type.
Key Findings
- 50 mg/kg PS-NPs (50 nm) administered daily for 35 days caused confirmed accumulation in mouse testicular tissue by Py-GC/MS and significant histological disruption of seminiferous tubules as scored by the Johnsen system
- Sperm density, motility, and viability were significantly reduced and sperm abnormality rates significantly increased in PS-NP-exposed mice versus water controls (n=10/group)
- In GC-2 spermatocytes, PS-NPs induced dose-dependent ferroptosis with elevated Fe²⁺, increased MDA, depleted GSH, upregulated lipid peroxidation (C11-BODIPY), and downregulated GPX4, SLC7A11, and FTH1
- Iron chelation with deferiprone (3.5 µM) and autophagy inhibition with 3-MA (10 mM) each significantly mitigated PS-NP-induced ferroptosis, confirming iron-dependent autophagic mechanism
- Cy5-PS-NP time-lapse imaging showed lysosomal accumulation followed by mitochondrial transfer within 12 hours, coinciding with elevated mitochondrial Fe²⁺ and ROS and structural mitochondrial damage
- CISD1 protein was significantly downregulated by PS-NPs; NCOA4 overexpression replicated ferroptotic effects; pioglitazone (5 µM) restored CISD1, reduced mitochondrial iron overload, and rescued cells from ferroptosis
- Serum testosterone and FSH were disrupted in PS-NP-exposed mice, indicating systemic reproductive endocrine impact beyond local testicular damage
Methodology
Twenty male KM mice (5 weeks, SPF) were randomized to PS-NP (50 mg/kg/day oral gavage, 35 days) or water control (n=10/group); testicular histology used Johnsen scoring and sperm analysis used CASA. In vitro work used authenticated GC-2 mouse spermatocyte cells exposed to 50–200 µg/mL PS-NPs, with mechanistic interventions including deferiprone, 3-MA, pioglitazone, and NCOA4 overexpression. PS-NP subcellular trafficking was tracked via Cy5-labeled particles and fluorescence microscopy; ferroptosis was quantified by Fe²⁺ assays, C11-BODIPY lipid peroxidation, GSH/MDA kits, flow cytometry, Western blot, and electron microscopy. Statistical methods and specific p-values were not fully reported in the available text excerpt.
Study Limitations
The study used a single PS-NP size (50 nm) and polymer type (polystyrene), limiting generalizability to the full spectrum of environmental nanoplastic exposures. The mouse model and GC-2 cell line may not fully recapitulate human spermatogenesis, and specific effect sizes with p-values for most outcomes were not comprehensively reported. No conflicts of interest were declared, though the dose selection and model conditions represent idealized laboratory exposures rather than chronic real-world mixed exposures.
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